- Volume 146, Issue 10, 2000

Trichomonas vaginalis grown for 16 h in the presence of [14C]spermine formed a high intracellular pool of [14C]spermidine and a small but detectable pool of [14C]putrescine. When [3H]putrescine was added to the growth medium, a large intracellular pool of [3H]putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T. vaginalis. Spermidine:spermineN 1-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine. Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction. In the presence of saturating substrate, the trichomonad SSAT had an activity of 0·39±0·09 nmol min−1 (mg protein)−1 (the mean of five analyses) and an apparent K m for spermine of 1·7 μM. The enzyme was competitively inhibited by di(ethyl)norspermine with a K i of 28 μM. Growth studies indicated that 50 μM di(ethyl)norspermine caused a 68% and 84% reduction in the intracellular concentrations of spermidine and spermine, respectively. The trichomonad polyamine oxidase required FAD as a cofactor and had an apparent K m of 6·0 μM forN 1-acetylspermine. The potential of bis(alkyl) polyamine analogues as antitrichomonad agents is discussed.

Intracellular pH (pHi) is an essential parameter in the regulation of intracellular processes. Thus, its measurement might provide clues regarding the physiological state of cells cultivated in vitro. pHi of the filamentous, pristinamycin-producing Streptomyces pristinaespiralis was determined by epifluorescence microscopy and image analysis using the pH-sensitive fluorescent probe BCECF-AM [2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. Staining cell culture samples (OD660=1) of S. pristinaespiralis with 20 μM BCECF at 28 °C for 30 min yielded a green/red fluorescence ratio (R 527/600) that correlated with the pHi of the cells for values ranging from 6·5 to 8·5. When S. pristinaespiralis was cultivated in pristinamycin-producing conditions (in batch mode, with a constant external pH of 6·8), the measured pHi varied between 6·3 and 8·7. In fact, pHi correlated with the excretion of pristinamycins and glucose consumption during the production process.

Until the discovery of the V ibrio c holerae repeat (VCR), the gene capture and expression systems termed integrons had been typically associated with antibiotic-resistance gene cassettes with usually less than five genes in an array. A method is described for the cloning of the ends of large cassette arrays. Conserved restriction sites within VCRs facilitated the mapping by Southern hybridization and cloning of the 5’ end of the VCR array, and using appropriate fragments it was possible to develop a physical map of the region of the V. cholerae chromosome. Sequence determination of the predicted beginning of this region revealed intI4, a member of the integron family of integrases. Comparison of these sequences from El Tor, Classical and serotype O134 V. cholerae strains identified the 3’ end of the attI site, thereby defining the class 4 integron in one of the V. cholerae chromosomes, and providing the first evidence for integron-like site-specific recombination within V. cholerae. Conduction assays demonstrated IntI1-mediated recombination between VCRs. Restriction mapping places the sequences of intI4 and 26 VCR gene cassettes in arrays within a 120 kb region of the V. cholerae O1 strain 569B genome. This region contains an estimated 150 VCR gene cassettes, dwarfing previously described arrays. Southern analysis of genomic DNA from strains of Vibrio anguillarum, Vibrio mimicus and a number of V. cholerae serotypes revealed fragments that hybridized with VCR-specific probes but showed a high degree of restriction fragment length polymorphism. These data facilitate the identification of part of a new class 5 integron from V. mimicus.

The promoter of the Paracoccus denitrificans nitric oxide reductase operon (norCBQDEF) has been characterized by primer extension and deletion analysis. A major transcript that is detectable only in anaerobically grown cells initiates 43·5 bp downstream of the centre of a putative binding site for the transcription factor NNR (nitrite and nitric oxide reductase regulator, which is known to regulate nor expression). A minor transcript initiates 121 bp upstream of the major transcript and is detectable in cells grown aerobically or anaerobically. Deletion derivatives of the nor promoter region were constructed and analysed in vivo using transcriptional fusions to the reporter gene lacZ. Expression patterns from promoter deletions in a wild-type strain and an nnr mutant confirmed that the minor transcript is NNR independent, and makes a small contribution to nor expression under both aerobic and anaerobic growth conditions. A deletion derivative truncated to within 7 bp of the putative NNR-binding site showed a near wild-type response to anaerobic growth, showing that no upstream DNA sequences are required for activation of the major promoter. Site-directed mutagenesis of the putative NNR-binding site confirmed that this is the major cis-acting sequence mediating the anaerobic inducibility of nor expression.

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli σ70 promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The −10, −16, −42 and −52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the −10, −16 and −35 regions, but also that of position −33, and even more importantly, maintenance of the upstream promoter element at position −42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.

Normally glucosamine (GlcN) is not a substrate for EIICBGlc of the glucose phosphotransferase system (PTS), encoded by ptsG, but it is transported by the mannose (Man) PTS, encoded by manXYZ. A mutation, umgC, has been described in Escherichia coli which allows a strain mutated in the Man PTS to grow on GlcN. The umgC mutation was mapped to the ptsG region and was proposed to make ptsG expression constitutive. Transcription of ptsG is regulated by the repressor Mlc so that mutations in mlc enhance the expression of ptsG. An mlc mutation, however, is not sufficient to allow good growth on GlcN, unlike the umgC mutation. The umgC mutation is shown to enhance expression of ptsG even in the absence of any PTS sugar transport, but the increase is greater in the presence of GlcN or Man. The umgC mutation also increases expression of the ptsHI and manXYZ operons, which are both regulated by Mlc. The umgC mutation was sequenced and two mutations were found: one, G176D, within the IIC membrane domain and the second, E472K, within the soluble IIB domain of PtsG. The cloned UmgC allele shows the enhanced transport and regulatory characteristics of the chromosomal mutation. Analysis of the two mutations present individually on plasmids shows that the IIC mutation is responsible for both the effect on sugar specificity and regulation.

Antisense peptide nucleic acids (PNAs) can inhibit Escherichia coli gene expression and cell growth through sequence-specific RNA binding, and this opens possibilities for novel anti-infective agents and tools for microbial functional genomics. However, the cellular effects of PNAs are limited relative to effects in cell extracts, presumably because of cell barrier components such as the outer-membrane lipopolysaccharide (LPS) layer or drug efflux pumps, both of which function to exclude antibiotics and other foreign molecules. To evaluate the importance of such cellular factors on PNA effects, the authors developed a positive assay for antisense inhibition by targeting the lac operon repressor and compared PNA susceptibilities in mutant and wild-type E. coli by assessing lacZ induction. Strains with defective LPS (AS19 and D22) were more permeable to the antibiotic nitrocefin and more susceptible to PNA than the wild-type. Also, PNA potency was improved in wild-type cells grown in the presence of certain cell-wall-permeabilizing agents. In contrast, the activities of the Acr and Emr drug efflux pumps were not found to affect PNA susceptibility. The results show that the LPS layer is a major barrier against cell entry, but PNAs that can enter E. coli are likely to remain active inside cells.

In the past it has been reproducibly demonstrated that 37 °C-grown DBY747 yeast cells have 29% more unsaturated fatty acids and a 3 °C higher maximal heat shock response (HSR) than their 25 °C counterparts. Suddenly the HSR and lipid profiles of cells grown at 25 °C and 37 °C became indistinguishable from one another. This paper reports an aberrantly high level of unsaturated fatty acids and an abnormally insensitive HSR in cells grown at 25 °C in yeast nitrogen base (YNB) that has been reconstituted from dehydrated medium packaged in ’new’ plastic containers. Effective even at a 1:600 dilution of reconstituted medium in laboratory-made YNB, the ’active ingredient’ was identified using a combination of HPLC and mass spectroscopy as dioctyl phthalate (a plasticising agent). Furthermore, the same levels of increase in the percentage of unsaturated fatty acids and decrease in the sensitivity of HSR were found in cells grown in laboratory-made YNB that contained as little as 36 μM pure dioctyl phthalate. This compound nevertheless failed to elicit an observable effect on cellular growth rate at levels up to and including 144 μM. These results suggest that dioctyl phthalate causes yeast cells to accumulate high levels of unsaturated fatty acids with a concomitant decrease in the sensitivity of the HSR, without compromising overall cellular function. They also support earlier work that suggested that the HSR is exquisitely sensitive to the level of unsaturated fatty acids present in yeast cells.

The Aspergillus nidulans cysA gene was cloned by functional complementation of the cysA1 mutation that impairs the synthesis of O-acetylserine. The molecular nature of cysA1 and cysA103 alleles was characterized; a nucleotide substitution and a frame shift were found in the former and a deletion mutation in the latter. The CYSA protein is 525 amino acids long and is encoded by an uninterrupted open reading frame. Expression of the cysA gene appears not to be regulated by sulfur, carbon and nitrogen sources. Protein sequence analysis reveals extensive similarity to homoserine O-acetyltransferases, particularly the bacterial ones, and no homology with known serine O-acetyltransferases. The authors propose that the CYSA protein is analogous to serine O-acetyltransferases, i.e. it catalyses the same reaction but has an independent evolutionary origin.

Using the complete genome sequence of Pseudomonas aeruginosa PAO1, sequenced by the Pseudomonas Genome Project (ftp://ftp.pseudomonas.com/data/pacontigs.121599), a genome database (http://pseudomonas.bit.uq.edu.au/) has been developed containing information on more than 95% of all ORFs in Pseudomonas aeruginosa. The database is searchable by a variety of means, including gene name, position, keyword, sequence similarity and Pfam domain. Automated and manual annotation, nucleotide and peptide sequences, Pfam and SMART domains (where available), Medline and GenBank links and a scrollable, graphical representation of the surrounding genomic landscape are available for each ORF. Using the database has revealed, among other things, that P. aeruginosa contains four chemotaxis systems, two novel general secretion pathways, at least three loci encoding F17-like thin fimbriae, six novel filamentous haemagglutinin-like genes, a number of unusual composite genetic loci related to vgr/Rhs elements in Escherichia coli, a number of fix-like genes encoding a micro-oxic respiration system, novel biosynthetic pathways and 38 genes containing domains of unknown function (DUF1/DUF2). It is anticipated that this database will be a useful bioinformatic tool for the Pseudomonas community that will continue to evolve.

The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics. Restriction mapping uncovered an 8·9 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus. These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P. aeruginosa clone C isolates. The breakpoints of the deletion in clone K and the attB–attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3’ end of the lysine tRNA structural genes (att site). pKLK106 sequentially recombined with either of the two tRNALys genes in clone K isolates. The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome; the att site of the phnAB–oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements. The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA. The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.

It was previously shown that most Pseudomonas syringae strains contain one or more plasmids with cross-hybridizing replication regions and other areas of homology, and these plasmids were designated the pPT23A-like family. The majority of these plasmids encode genes conferring epiphytic fitness or resistance to antibacterial compounds and those investigated in this study are essential for pathogenicity or increased virulence. The phylogeny of 14 pPT23A-like plasmids from five P. syringae pathovars was studied by comparing a fragment of the sequence of their repA genes (encoding a replicase essential for replication). In the phylogenetic tree obtained, four groups (≤88·8% identity between their members) could be identified. The first group contained the plasmids from three P. syringae pv. tomato strains, a P. syringae pv. apii strain and five out of the seven P. syringae pv. syringae strains, with identity ranging between 88·8 and 100%. The clustering of the pv. syringae strains did not reflect host specialization or previously reported phylogenetic relationships. The second group contained the plasmids from two strains of pv. glycinea and pv. tomato (95·5% identity), and it also included the previously sequenced replicon of a pathogenicity plasmid from P. syringae pv. phaseolicola. The plasmids from the remaining two pv. syringae strains were distantly related to the other plasmid sequences. Hybridization experiments using different genes or transposable elements previously described as plasmid-borne in P. syringae, showed that the gene content of highly related plasmids could be dissimilar, suggesting the occurrence of major plasmid reorganizations. Additionally, the phylogeny of the different native plasmids did not always correlate with the phylogeny of their harbouring strains, as determined by the analysis of extragenic repetitive consensus (ERIC) and arbitrarily primed PCR (AP-PCR) products. Collectively, these results suggest that pPT23A-like plasmids were, in most cases, acquired early during evolution.